Botulinum neurotoxin type B (BoNT/B) is part of a family of seven structurally related proteins (botulinum toxins A to G) produced by various strains of the anaerobic bacillus “clostridium botulinum ”. The two most commonly encountered forms are botulinum toxins type A and B. Botulinum neurotoxins are the most powerful known toxins, with lethal dose 50s in mice of the order of 0.1 to 0.3 ng/kg. They act on the peripheral nervous system of humans and of various animal species, inducing “botulism”, which is characterized by flaccid paralysis of the skeletal muscles, leading to death.
The major form of poisoning with these toxins is due to ingestion of contaminated food. These proteins may constitute a potential biological weapon since they are easy to produce. Finally, for several years, botulinum toxins type A and also type B have also been used for therapeutic applications, in the context of dystonias and of motoneuronal hyperactivity, such as strabismus or blepharospasm.
Botulinum neurotoxins consist of two subunits: a heavy chain (˜100 kDA) linked to a light chain (˜50 kDA) by a disulfide bridge. The heavy chain is involved in binding of the toxin to the nerve ending, in internalization and then in translocation of the light chain in the cytosol. The light chain is responsible for the toxicity of the protein by inhibiting Ca2+−dependent release of acetylcholine. The light chain can only express its toxicity when it is separated (reduction of the disulfide bridge) from the heavy chain, but it is not capable of penetrating alone into nerve endings (Montecucco et al. (1994) FEBS Lett. 346, 92–98).
The toxicity of the light chain of these toxins is due to its peptidase activity. Specifically, botulinum toxins belong to the zinc metallopeptidase family, and most particularly to the subfamily of zincins which contain the consensus sequence HExxH (Schiavo et al. (1992) J. Biol. Chem. 267(33), 23479–23483). They very specifically cleave neuronal proteins involved in neurotransmitter exocytosis. Thus, syntaxin and SNAP 25 are degraded by botulinum toxins A, C and E, whereas synaptobrevin (VAMP) is cleaved by tetanus toxin and botulinum toxins B, D, F and G. It is important to note that the site of cleavage of these proteins by the toxins is different, except for BoNT/B and tetanus toxin, which cleave synaptobrevin at the Q76—F77 bond.
The most effective approach for combating the harmful effects of BoNT/B, either in the course of declared botulism or in the course of therapeutic contraindications, is the development of selective inhibitors with high affinity for its metallopeptidase activity, which is responsible for its toxicity. However, the identification of such inhibitors requires a simple and automatable test for demonstrating BoNT/B activity, allowing a large number of assays.
Now, the tests currently available are not entirely satisfactory. They are either long or relatively insensitive and/or unsuitable for an implementation such as a high throughput screening method.
Currently, the most sensitive method for detecting botulinum toxins is based on an in vivo assay in mice (Kautter & Salomon (1976) J. Assoc. Anal. Chem. 60, 541–545). This assay makes it possible to detect from 5 to 10 pg of toxin, but the response time is much too long (3–4 days), the serotype of the toxin is not known and, finally, animal experimentation is very controversial. Assays on cell lines have been attempted (DeWaart et al. (1972) Zentralblatt für Bacteriologie 222, 96–114), but the sensitivity thereof is too low. Immunoassays have also been proposed, but none shows sufficient sensitivity, even after amplification of the response (Stanley et al. (1985) J. Immunol. Methods 83, 89–95; Doellgast et al. (1993) J. Clin. Microbiol. 31, 2402–2409). A relatively sensitive calorimetric test has been proposed (Szilaggi et al. (2000) Toxicon 35 381–389) for BoNT/B, but it involves a succession of steps which make it non-automatable.
Other assays currently developed are based on the use of the endopeptidase activity of these toxins (Hallis et al. (1996) J. Clin. Microbiol. 34, 1934–1938). This involves cleavage, by BoNT/B, of a fragment of synaptobrevin immobilized on a solid support, and detection, with a specific antibody, of the N-terminal end newly created by cleavage and which remains on the resin. A final protocol consists in immobilizing the toxin on an affinity column. When passed over this column, the substrate is cleaved and the fragment is recognized by an antibody [Witcome et al. (1999) Applied and environmental microbiology 65 3787–3792]. However, these assays have not yet been optimized and are very difficult to automate.